The present invention relates to CEA binding polypeptides and compositions for detection and treatment of cancer. More particularly, the invention relates to materials useful for and methods of detecting, imaging, localizing, and targeting tumors exhibiting CEA. The invention provides binding polypeptides capable of associating specifically with CEA and of distinguishing between CEA and known cross-reactive antigens, such as NCA (non-specific cross-reacting antigen). Such binding polypeptides are useful for the detection, imaging, localization, and targeting of CEA-containing tissues or solutions, e.g., by radioimaging, magnetic resonance imaging, or x-ray imaging, and are also useful in the diagnosis and treatment of cancers associated with CEA.
Carcinoembryonic antigen, or CEA, is a complex immunoreactive glycoprotein with a molecular weight of 180,000 found in adenocarcinomas of endodermally derived digestive system epithelia and fetal colon. Tumor cells at many sites, including colon, breast, lung, cervix, ovary, stomach, bladder, pancreas and esophagus express large amounts of carcinoembryonic antigen and/or the closely related immunoglobulin supergene family member, nonspecific cross-reactive antigen, or NCA, on their surfaces. The expression of these glycoproteins, especially CEA, in normal cells is very limited in mature individuals (as opposed to prenatal infants), and this antigen has been used as a target in immunoassays for diagnosis and for serially monitoring cancer patients for recurrent disease or response to therapy. (See, Mach et al., Immun. Today, 2:239, 1981; Berche et al., Br. Med. J., 285: 1447, 1982.) Anti-CEA antibodies also have been proposed for cancer therapy and for use in forming immunoconjugates which in turn can be used in cancer therapy. (See, e.g., Buchegger et al., U.S. Pat. No. 5,047,507 (1991); Osbourne et al. U.S. Pat. No. 5,872,215 (1999).)
CEA was as first described by Gold and Freedman, J. Exp. Med., 121: 439, 1965, and has now been completely sequenced and characterized (see, Beauchemin et al., Mol. Cell. Biol., 7:3221-30, 1987; WO 95/06067). CEA has a domain structure of N-A1-B1-A2-B2-A3-B3-GPI where GPI is a glycophosphatidylinositol membrane anchor. A significant degree of sequence homology exists between the domains of CEA and other members of the immunoglobulin supergene family, and immunological cross-reactivity between CEA and as many as sixteen other homologous antigens, such as NCA and biliary glycoprotein-1 (BGP-1), has been reported.
One of the major drawbacks of the use of anti-CEA antibodies for clinical purposes has been the cross-reactivity of these antibodies with some apparently normal adult tissues. Previous studies have shown that most conventional hyperimmune antisera raised against different immunogenic forms of CEA cross-react with CEA-related antigens found in normal colonic mucosa, spleen, liver, lung, sweat glands, polymorphonuclear leukocytes and monocytes of normal individuals, as well as many different types of carcinomas.
Accordingly, there is a great need for binding moieties that bind to CEA but do not cross-react with other antigens such as NCA. This and other objects are accomplished herein with the discovery of novel peptide binders of CEA.
The present invention addresses the need for improved materials and methods for detecting, localizing, measuring and treating CEA-expressing cells by providing a group of non-naturally occurring polypeptides that bind specifically to CEA. Appropriate labeling of such polypeptides provides detectable imaging agents that bind at high concentration to a CEA-expressing tumor, providing excellent tumor-specific imaging agents. Conjugation or fusion of such polypeptides with effective agents such as cytokines, chemotherapeutic agents, radionuclides or other cancer therapeutics produce conjugates that can be used for cancer therapy, i.e., by causing the conjugate to target the site of a tumor that is producing CEA. Recombinant baeteriophage displaying the CEA-binding polypeptides of the invention have been identified and isolated, and such phage products are also valuable reagents for effective detection and diagnosis of cancers associated with the expression of CEA in cells and tissues. The CEA binding moieties of the instant invention can be used in the detection, diagnosis, and therapy of such CEA-related disorders.
This invention pertains to CEA binding moieties. Binding moieties according to this invention are useful in any application where binding, detecting or isolating CEA or its fragments is advantageous. A particularly advantageous use of the binding moieties disclosed herein is in a method of imaging cells or tissues expressing CEA in vivo. The method entails the use of CEA specific binding moieties according to the invention for detecting CEA-expressing cells, where the binding moieties have been detectably labeled for use as imaging agents, including magnetic resonance imaging (MRI) contrast agents, x-ray imaging agents, radiopharmaceutical imaging agents, ultrasound imaging agents. and optical imaging agents.
Preferred CEA binding moieties according to the invention are isolated synthetic polypeptides having a high affinity for CEA. This invention provides a new class of CEA binding polypeptides having an amino acid sequence comprising:
X1-X2-X3-Cys-X4-X5-X6-X7X8-X9-X10-X11-Cys-X12-X13-X14xe2x80x83xe2x80x83(SEQ ID NO:1),
wherein
X1 is Asn, Asp, or is absent;
X2 is Trp;
X3 is Asp, Phe, or Val;
X4 is Asn, Glu, or Met;
X5 is Asn, Leu, Met or Phe;
X6 is Asp, Gly, Ile, Lys, Phe or Thr;
X7 is Ala, Gin, Gly, Lys, or Thr;
X8 is Arg, Asn, Asp, Glu, or Gly;
X9 is Gln, Gly, or Leu;
X10 is Ala, Trp or Tyr;
X11 is Ala, Gly, His, Phe, Thr, or Val;
X12 is Asn, Gln, Phe, Ser or Val;
X13 is Arg, Leu, Pro or Ser; and
X14 is Leu, Ser, Trp, or Tyr;
and wherein said polypeptide has the ability to bind CEA. Said polypeptide may have additional amino acids attached at either end. Peptides having a serine at the N-terminus (before X1) are preferred embodiments.
Preferred CEA binding polypeptides of the above formula will have the amino acid sequence:
X1-Trp-Val-Cys-Glu-X5-X6-Lys-X8-Gln-Trp-X11-Cys Asn-X13-X14xe2x80x83xe2x80x83(SEQ ID NO:2),
wherein
X1 is Asn or Asp;
X5 is Asn, Leu, Met or Phe;
XH is Asp, Gly, Ile, Lys, Phe or Thr;
X5 is Arg, Asn. Asp, Glu, Gly or Trp;
X11 is Ala, Gly, His, Phe, Thr, Tyr or Val;
X13 is Arg, Leu, Pro or Ser; and
X14 is Leu, Ser, Trp or Tyr;
In particular, a stable binding loop having a high affinity for CEA is disclosed, having the formula:
Cys-X4-X5-X6-X7-X8-X9-X10-X11-Cysxe2x80x83xe2x80x83(SEQ ID NO: 3),
wherein
X4 is Asn, Glu, or Met;
X5 is Asn, Leu, Met or Phe;
X6 is Asp, Gly, Ile, Lys, Phe or Thr;
X7 is Ala, Gln, Gly, Lys, or Thr;
X8 is Arg, Asn, Asp, Glu, or Gly;
X9 is Gln, Gly, or Leu;
X10 is Ala, Trp or Tyr;
X11 is Ala, Gly, His, Phe, Thr, or Val;
and wherein it is preferred that
X4 is Glu;
X5 is Asn, Leu, Met or Phe;
X6 is Asp, Gly, Ile, Lys, Phe or Thr;
X7 is Lys;
X8 is Arg, Asn, Asp, Glu, or Gly;
X9 is Gln:
X10 is Trp; and
X11 is Ala, Gly, His, Phe, Thr, or Val.
Preferred polypeptides according to the invention comprise an amino acid sequence:
Asn-Trp-Val-Cys-Asn-Leu-Phe-Lys-Asn-Gln-Trp-Phe-Cys-Asn-Ser-Tyrxe2x80x83xe2x80x83(SFQ ID NO:4)(TN 10/9-G08),
Asp-Trp-Val-Cys-Glu-Asn-Lys-Lys-Asp-Gln-Trp-Thr-Cys-Asn-Leu-Leuxe2x80x83xe2x80x83(SEQ ID NO:5)(TN10/9-A07),
xe2x80x83Asn-Trp-Asp-Cys-Met-Phe-Gly-Ala-Glu-Gly-Trp-Ala-Cys-Ser-Pro-Trpxe2x80x83xe2x80x83(SEQ ID NO:6)(TN10/9-E01),
Asp-Trp-Val-Cys-Glu-Lys-Thr-Thr-Gly Gly-Tyr-Val-Cys-Gln-Pro-Leuxe2x80x83xe2x80x83(SEQ ID NO:7)(TN10/9-B09),
Asn-Trp Phe-Cys-Glu-Met-Ile-Gly-Arg-Gln Trp-Gly-Cys-Val-Pro-Serxe2x80x83xe2x80x83(SEQ ID NO:8)(TN10/9-F11),
and
Asp-Trp-Val-Cys-Asn-Phe-Asp-Gln-Gly-Leu-Ala-His-Cys-Phe-Pro-Serxe2x80x83xe2x80x83(SEQ ID NO:9)(TN10/9-D04).
The most preferred CEA binding moieties according to the invention are isolated, synthetic polypeptides having a high affinity for CEA. This invention provides a new class of CEA binding polypeptides having an amino acid sequence comprising:
X1-X2-X3-Cys-X4-X5-X6-X7-X8-X9-X10-X11-Cys-X12-X13-X14xe2x80x83xe2x80x83(SEQ ID NO:1),
wherein
X1 is Asp, Asn, Ala, or Ile, with Asp most preferred;
X2 is Trp;
X3 is Val, Ile, Met, Tyr, Phe, Pro, or Asp, with Val most preferred;
X4 is Asn, Glu, or Asp, with Asn and Glu most preferred;
X5 is Leu, Phe, Tyr, Trp, Val, Met, Ile, or Asn, with Leu most preferred;
X6 is Phe, Leu, Asp, Glu, Ala, Ile, Lys, Asn, Ser, Val, Trp, or Tyr, with Phe most preferred;
X7 is Lys, Phe, Asp, Gly, Leu, Asn, or Trp, with Lys most preferred;
X8 is Asn, Pro, Phe, Gly, Asp, Ala, Ser, Glu, Gln, or Trp, with Asn most preferred;
X9 is Gln, or Lys, with Gln most preferred;
X10 is Trp;
X11 is Phe, Thr, Met, Ser, Ala, Asn, Val, His, Ile, Pro, Trp, or Tyr, with Phe most preferred;
X12 is Asn, Asp, Glu, Pro, Gln, or Ser, with Asn and Asp most preferred;
X13 is Val, Leu, Ile, Pro, Ala, Gln, Ser, Met, Glu, Thr, Lys, or Trp, with Val and Leu most preferred; and
X14 is Leu, Met, Val, Tyr, Ala, Ile, Trp llis, Pro, Gln, Glu, Phe, Lys, or Arg, with Leu most preferred.
The polypeptides listed in Table 5 (infra) are preferred embodiments of the present invention. Polypeptides 304A-12-H12 (SEQ ID NO:59), 304A-14-B02 (SEQ ID NO:74), 304A-14-A12 (SEQ ID NO:83), and 304A-15-E04 (SEQ ID NO:92) are especially preferred embodiments of the present invention.
Another aspect of the present invention relates to modifications of the foregoing polypeptides to provide CEA specific imaging agents, wherein the binding moieties are modified by radiolabeling, enzymatic labeling, or labeling with MR paramagnetic chelates; or wherein the binding moieties are incorporated in microparticles, ultrasound bubbles, microspheres, emulsions, or liposomes; or wherein the binding moieties are conjugated with optical dyes.
In another aspect of the present invention, methods for isolating CEA binding moieties are provided. Such methods will be useful for isolating additional reagents for detection, localization, quantification, and treatment of neoplastic disorders associated with upregulated CEA expression.
In another aspect of the invention, methods of diagnosing CEA-associated disorders and methods for localizing CEA-expressing cells or tissues, are provided, and methods for treating cancers indicated by increased CEA expression are provided.
In another aspect of the invention, therapeutic agents comprising a combination, conjugation or fusion of a anticancer drug or other therapeutic agent with a CEA binding moiety according to the invention are provided. Such compositions will be useful in the treatment of CEA-associated disorders and conditions.
In another aspect of the invention, recombinant baeteriophage displaying CEA binding polypeptides on their surfaces are also provided. Such phage are useful as screening reagents and reagents for detecting CEA.
Another aspect of the invention relates to forming molecules containing multiple CEA-binding moieties to increase the residence time of these molecules on CEA targets. These multimeric molecules can be altered to provide CEA specific imaging agents by radiolabeling, enzymatic labeling, or labeling with MR paramagnetic chelates or microparticles; ultrasound bubbles, microparticles, microspheres, emulsions, or liposomes; or optical dyes.
Another aspect of the invention relates to introducing DNA that encodes one or more CEA-binding moieties into the coat protein of a virus to cause the virus to hind and preferentially infect CEA-bearing cells. Such alteration will make the virus target CEA-expressing (tumor) cells.
These and other aspects of the present invention will become apparent with reference to the following detailed description.